Saccharomyces Cerevisiae ChromosomeⅦ Synthesis

In 2011, professor Jef Boeke from New York University(NYU) led to set up the "Synthetic Yeast Genome Project" referred to as "SC2.0" program. The universities and research institutes from United States, Britain, China, Hong Kong and India were gathered together to synthesize 16 chromosomes and a single tRNA chromosome of saccharomyces cerevisiae artificially.So far, the synthesis of chr09 R, chr06 L and chr03 has been completed by Johns Hopkins University(JHU), their achievements were published in "Nature" and "Science".The synthesis of chr06, chr12, chr05 is nearing an end. BGI is in charge of synthesizing chr02, chr07 and chr13, the fellowing verification test of chr02 in yeast is underway now.Synchr07 synthesis project belong to the extension subject of "Saccharomyces cerevisiae chromosomes synthesis project". BGI is in charge of synthesizing chr07 L and University of Edinburgh(UoE) is in charge of synthesizing chr07 R. Chr02 synthesis project basically solved the technical problems in DNA assembling and replacement,but there’s still a huge space to improvement in the aspects of assembly efficiency and accuracy. Moreover, we still need to explore the functions of gene groups and new phenotypes, after chromosome rearrangement introduced by SCRaMbLE.In this research, we design a 1015 kb “designer” chromosome 7 based on the native one. Using SegMan to cut chr07 into different length of DNA fragments,respectively termed as minichunk(2-3 kb), chunk(~10 kb) and megachunk(~50 kb).Minichunk splicing into chunk by Gibson assembly in vitro, electrophoresis identification and sequencing results showed all 42 chunks of chr07 L has been assembled in a right way. Homologous replacement happened between chunks through1 kb overlap in yeast, from both ends to the middle. There are total 14 rounds of replacement in chr07 L from megachunk A to N, the first 12 rounds has been completed until now, and we get a replacement strain SynA-L. PCR Taq identification indicated that the megachunk A to L of this strain are all syn type, but PGM sequencing found a A3- B5 tandem copies variation in the strain. Whereafter we will further complete the remaining replacement, and repair the variation.Chr07 has obvious characteristics, such as maltose metabolism genes enrichment,has a maximum number of tRNAs etc. The research of synthesis chr07 with itsfollowing work is a good point for systematic study in aspects of genes’ function,genome structural variation and evolution of species.